Orthopoxviruses are complex enveloped viruses having a diameter comprised between 200 and 300 nm that distinguish them principally by their unusual morphology, their large DNA genome and their cytoplasmic site of replication. The genome of several members of Orthopoxviruses, including the Copenhagen Vaccinia Virus (VV) strain (GOEBEL et al., 1990, Virol. 179, 247-266 and 517-563; JOHNSON et al., 1993, Virol. 196, 381-401) and the modified Vaccinia Virus Ankara (MVA) strain (ANTOINE et al., 1998, Virol. 244, 365-396), have been mapped and sequenced. VV has a double-stranded DNA genome of about 192 kb coding for about 200 proteins of which approximately 100 are involved in virus assembly. MVA is a highly attenuated Vaccinia Virus strain generated by more than 500 serial passages of the Ankara strain of Vaccinia Virus on chicken embryo fibroblasts (MAYR et al., 1975, Infection 3, 6-16). The MVA virus was deposited before Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N602 1-721. Determination of the complete sequence of the MVA genome and comparison with the Copenhagen VV genome allows the precise identification of the alterations which occurred in the viral genome and the definition of seven deletions (I to VII) and numerous mutations leading to fragmented ORFS (Open Reading Frame) (ANTOINE et al., 1998, Virology 244, 365-396).
The use of Orthopoxviruses as vectors for the development of recombinant live vaccines has been affected by safety concerns and regulations. Before using Orthopoxviruses for vaccination it is necessary to purify the viruses. Available Orthopoxviruses production methods comprise the replication of the virus in a cell line (e.g. HelaS3), in embryonated eggs or in Chicken Embryo Fibroblasts (CEFs). After the replication of the virus, the culture media is discard, the cells are lysed and the Orthopoxviruses released from the cells are purified by sucrose cushion centrifugation (KOTWAL and ABRAHAM; poxvirus growth, Purification and tittering in Vaccinia Virus and Poxvirology, 2004, 101-108, Humana Press Inc., Totowa; NJ; USA).
International patent application WO 07/147,528 describes a method for producing a wild type, an attenuated and/or a recombinant MVA with no targeted infection specificity, comprising preparing a culture of packaging cells (e.g. CEFs; cell lines), infecting said cell culture, culturing said infected cells, recovering the MVAs produced from the culture supernatant and/or the packaging cells, and further purifying the viruses by depth filtration, microfiltration and diafiltration. WO 07/147,528 specifies that when depth filtration, microfiltration and diafiltration are performed, the use of nucleases and more particularly, the use of Benzonase® as nuclease is not necessary.
International patent application WO 08/138,533 describes a method for the purification of biologically active vaccinia virus, comprising loading a solid-phase matrix to which a ligand is attached with a vaccinia virus contained in a liquid-phase culture, washing the matrix, and eluting the virus.
Despite the description of several methods for Orthopoxvirus production and purification, a need remains for alternative. The present invention provides such methods.